Cryopreserved primary cultures of golden Syrian hamster embryo cells were used as the source of target and feeder cells for establishing an in vitro carcinogenesis bioassay. The primary culture giving the best overall response in a pretest before freezing gave positive results in 20 consecutive experiments when retested with 3-methylcholanthrene after cryopreservation, indicating that pretested cryopreserved cultures can serve as a source of susceptible target cells in an in vitro carcinogenesis bioassay. Similarly prepared and cryopreserved cultures served satisfactorily as feeder cells. Susceptible positive cultures were used to test a large number of carcinogenic and non-carcinogenic chemicals in this system. The results showed a very high positive correlation (90.8%) between morphological transformation and the reported carcinogenic activity of the chemicals. Transformation was not observed when cells were tested with a few carcinogens that may not be metabolized to their active forms by early passage hamster embryo cells. N-2-acetylaminofluorene transformed cells only when tested in the presence of hamster liver microsomes. No false positive results were obtained when non-carcinogens were bioassayed, nor was spontaneous transformation observed in control cultures treated with medium alone, 0.2% dimethylsulfoxide or other solvents. Cultures derived from morphologically transformed colonies arising after treatment of cells with several known carcinogens were tumorigenic in vivo, confirming the correlation of morphological transformation with tumorigenicity and the validity of altered morphology as an in vitro criterion for carcinogenicity in vivo.

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Before exerting a carcinogenic o r mutagenic effect, many carcinogens must undergo metabolic activation. Liver S-9 preparations from rats treated with Aroclor 1 2 5 4 (AC) have been widely used to extend the ability of the Salmonella mutagenicity assay t o detect such nondirect-acting agents. We compared the mutagen-activating capabilities of liver S-9 preparations f r o m both Syrian golden hamsters and Sprague-Dawley rats. T h e comparison was made between S-9 fractions from untreated, and phenobarbital (PB)-and AC-treated animals of both sexes. Also compared was the effect of varying the amount of S-9 protein. T h e preparations from hamster liver were consistently more effective than preparations from rat liver for the activation of 2-acetylaminofluorene (AAF), 2-aminoanthracene (AA) (except for ACinduced preparations), 3-methylcholanthrene (MCA), and diethylnitrosamine (DEN) t o their mutagenic forms. Induced preparations from rats were more active than those from hamsters when using benzo(a)pyrene (BP), while with uninduced preparations, the opposite was true.There was also a relationship between the a m o u n t of S-9 protein and the number of revertant colonies obtained. With the aromatic aniines ( A A F and AA), a partial inhibition of mutagenicity occurred at the highest protein concentrations tested. With BP, a n inverse relationship was observed between protein concentration and the number of revertants occurring in the presence of the preparation from PB-and AC-induced male and female rats.T h e relative order of activities of aryl hydrocarbon hydroxylase (AHH) and BP-4,Sepoxide hydrase (EH) in the preparations was AC-induced > PBllie abbreviations used are: AC.

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Summary.-Transformation of primary hamster embryo cells was investigated using 3-methylcholanthrene (MCA), a combination of MCA and 12-0-tetradecanoylphorbol-13-acetate (TPA), and initiation with MCA or dibenz(a,h)anthracene (DBA) followed by promotion with TPA. Evidence for transformation was (a) abnormal cellular morphology, (b) increased lifespan, (c) growth in soft agar, and (d) tumour induction by s.c. inoculation into suckling hamsters.Cells treated with either MCA or MCA+TPA showed the same latent period to morphological transformation, although their tumorigenic potential varied. Cells did not form tumours when TPA was administered 7 days after treatment with either MCA or DBA. However, when administration of TPA was delayed to 27 days after treatment with a transforming dose of MCA or a subthreshold dose of DBA, the cells transformed and produced tumours in hamsters.Our results show that TPA may act as an inhibitor or promoter, depending on the length of time between treatment of the hamster embryo cells with the carcinogen and administration of the TPA. It appears that treatment of cells with TPA before the initiating event is complete inhibits or delays the development of their ability to induce tumours in animals or grow in soft agar. However, with a sufficient interval between the application of the initiating carcinogen and the promoter, transformation occurs, and the ability of cells treated with subthreshold doses of DBA to form tumours is enhanced.